PROJECT SUMMARY/ABSTRACT Sarcomeres are specialized cytoskeletal structures in cardiomyocytes (CMs) that generate the force driving the heart contraction. Mutations in genes coding sarcomere components, or ?sarcomere mutations?, often cause inherited cardiomyopathies including most cases of inherited dilated cardiomyopathy (DCM). Sarcomere mutations are believed to impair CM contraction, thereby causing the contractile defects in DCM hearts. How- ever, whether other aspects of sarcomere function contribute to DCM pathogenesis has been incompletely studied. This knowledge gap prevents comprehensive understanding of DCM pathogenesis and limits the de- velopment of effective therapeutic strategies. This research proposal tests the overarching hypothesis that sarcomeres regulate signal transduction, which contributes to the pathogenesis of DCM. Specifically, Dr. Guo will dissect the mechanisms by which ac- tinin-2 (ACTN2) mutations perturb serum response factor (SRF) signaling through SRF cofactors MKL1/2, and the contribution of this pathway to DCM. This hypothesis will be tested through three Specific Aims. Aim 1 will study whether ACTN2 activates MKL-SRF signaling by promoting actin polymerization in CMs. Aim 2 will de- termine if then ACTN2-MKL1/2 pathway regulates transcription in CMs by affecting SRF-chromatin binding. Aim 3 will test the hypothesis that ACTN2-MKL-SRF signaling contributes to the pathogenesis of DCM caused by ACTN2 missense mutations. This project will reveal novel molecular insights about DCM pathogenesis. These insights may lead to new therapeutic approaches to treat DCM. This project is built upon state-of-the-art technologies such as adeno-associated virus (AAV)-delivered CRISPR/Cas9-based somatic mutagenesis and cardiac genetic mosaics analysis that were co-developed by Dr. Guo. K99/R00 will further sponsor training in induced pluripotent stem cells (hiPSCs), genome editing, sin- gle-cell transcriptomics and engineered heart tissues, which together with Dr. Guo?s past expertise will estab- lish a powerful and comprehensive technical platform for successful independent research. This grant will also support Dr. Guo?s education from a mentorship team of world-class scientists with relevant expertise and facili- tate Dr. Guo?s career development activities as he transitions toward independence.